Expression Of Dkk-3 And β-catenin In SD Rat’ Buccal Mucosal Carcinogenesis

Objective:Oral squamous cell carcinoma (OSCC) is the highest incidence of malignant tumors in the oral malignancies, mostly occurrs in middle ages of male patients. It has high degree of malignancy which often affected in the tongue, buccal, palate, gingival and often metastasized to the regional lymph nodes, advanced cancer may occur distant metastases. We are generally believed OSCC multi evolved from oral precancerous lesions and experienced the development process of the normal â†' precancerous lesions â†' carcinoma in situ â†' invasive carcinoma in turn. The most majority of OSCC have clear precancerous lesion stage that we can early detection, early diagnosis and early treatment, the 5-year survival rates of OSCC patients can also be improved. The medical science and technology has been rapid development in 21st century, more and more medical researchers are dedicated to explore tumor markers and make them for the diagnosis of the tumor early stages which has a unique role for the early detection of tumors and diagnosis of tumors. Wnt/β-catenin signaling pathway has been proved highly conserved on evolution and plays a key role in control embryonic development, abnormal activation of the pathways involved in the pathogenesis of many human cancers. Wnt/β-catenin signaling pathway is regulated by antagonist or agonist, many studies have shown that antagonists can inhibit the accumulation of free β-catenin in the cytoplasm which can transfer into the nucleus to activate proto-oncogenes, thereby they can inhibit the occurrence and development of tumor. Some researchers found that the tumor suppressor gene of Dkk-3 can inhibit the Wnt/β-catenin signaling pathway by block the large concentration of β-catenin in the cytoplasm, so that β-catenin can not transfer into the nucleus to start the proto-oncogene of downstream. In this study we use SD,s cheek tissue by they drinking the water added with 4NQ0 to induced oral carcinogenesis in Orofacial Reconstruction and Regeneration Lab, Dept. of Orthodontics in prophase, pathological diagnosis cheek tissue during carcinogenesis By HE staining and immunohistochemical to detect Dkk-3 and β-catenin protein expression of various stages, we also explore the correlation between Dkk-3 and β-catenin in OSCC development to find new experimental evidence for the prevention and treatment of OSCC. Methods:60 male SD rats(died 2), the experimental group (50 rats) drinking water containing 40 ppm of 4NQO,10 to drink ordinary tap water as negative control group. We killed 10 experimental rats at the start of the 9th week of each interval of 4 weeks after weekend at random. The left ten controlled rats which were feed without 4NQO would be killed at the end of animal experiment. We cut the oral mucosa to embedded in paraffin and slices,the sections were stained with HE and two senior pathology teacher classified those sections by pathological diagnosis and classification. The expression of Dkk-3 and β-catenin protein were detected by immunohistochemistry (SP) in each sample and analyzed the results by Statistical analysis (SPSS 20.0). Results:1. HE staining pathological grade 16 cases of normal mucosa,11 cases of mild dysplasia,12 cases of moderate dysplasia,10 cases of severe dysplasia and 9 cases of squamous cell carcinoma. 2.β-catenin immunohistochemical staining results showed:the expression of β-catenin in membrane was mainly located in the buccal mucosa epithelial cells on normal, with the increase of the degree of epithelial dysplasia the expression of β-catenin appeared in cytoplasmic; the expression of β-catenin mostly appeared in cytoplasm and small in nuclear membrane in severe dysplasia group; the β-catenin was only expressed in the nucleus of squamous cell carcinoma group. Ectopic expression rate of β-catenin in normal buccal mucosa was 18.75%; ectopic expression rate of β-catenin in mild dysplasia group and moderate dysplasia group were 45.45% and 58.33%; ectopic expression rate of β-catenin in sebere dysplasia was 80%; buccal cancer group of β-catenin ectopic expression rate was 88.89%. It has statistics significance of ectopic expression rate of β-catenin in normal buccal mucosa group to buccal cancer group in total (P<0.05); spearman analysis tells us ectopic expression rate of (3-catenin and histological grade has positive correlation (rs=0.386, P< 0.05).3. Dkk-3 immunohistochemical staining showed:the visible expression of a large number of brown in normal buccal mucosa epithelial cells, cytoplasm in SD rats, especially in spinous layer; the expression of Dkk-3 in the cytoplasm was decreased while the degree of dysplasia of the epithelial cells was increased, and the expression of Dkk-3 begen to appeared in nucleus; there was almost no expression of Dkk-3 in squamous cell carcinoma. The positive expression rate of Dkk-3 in normal buccal mucosa was 81.25%; the positive expression rate of Dkk-3 in mild buccal mucosa dysplasia group, moderate buccal mucosa dysplasia group, severe buccal mucosa dysplasia group were accounted for 63.67%,50%,20%; in buccal carcinoma the positive expression rate of Dkk-3 was only 11.11%. The chi-square tells us the differential of positive expression of Dkk-3 in normal buccal mucosa group to buccal career group has a statistically significant (P<0.01);Spearman analysis shows us the differential of positive expression of Dkk-3 and pathological grade has negative correlation(rs=-0.521, p<0.05).4. The difference of expression of Dkk-3 and P-catenin in the differential expression of normal buccal mucosa to carcinoma displays that ectopic expression rate of β-catenin was increased while the positive expression rate of Dkk-3 was decreased with the degree of dysplasia increase, statistical analysis the different expression of them shows a significant negative correlation(rs=-0.515,P< 0.05). Conclusions:1 The ectopic expression rate of β-catenin is increased with the severity of pathological in the squamous epithelial from SD rats’normal buccal mucosa to cancer process; 2 The positive expression of Dkk-3 in epithelial cells is decreased from SD rats’ normal buccal mucosa to cancer process; 3 There is negative correlation between Dkk-3 and β-catenin from the dynamic process of SD rats in normal buccal mucosa to carcinoma of buccal, this change of the expression of Dkk-3 and P-catenin suggests that Dkk-3 may play a role as a tumor suppressor to inhibit the ectopic expression of β-catenin., thus it can inhibit abnormal activation of Wnt/β-catenin signaling pathway.